Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 expression is increased in the nigrostriatal system of a monkey model of PD induced by α-syn PFFs injection. (A) Clinical rating score of monkeys after stereotactic injection of 600 μg of α-syn PFFs or an equal volume of normal saline for 4 months. (B) Immunohistochemistry detection of TH in the STR (left) and SN (right). The TH signal intensity in the STR and the number of TH-positive neurons in the SN were lower in the PFF group. The black arrow heads indicate typical TH-positive neurons. Scale bars: 100 μm. (C) Quantitative immunohistochemistry density analysis of TH in the STR. (D) Quantitation of the ratio of TH-positive neurons in the SN in the PFF group compared with the Sham group. (E) Western blot analysis of TH, LRP1, and α-syn expression levels in the monkey STR. (F–H) Densitometric analysis of TH (F), LRP1 (G), and α-syn (H) expression levels in the STR. (I) Western blot analysis of TH, LRP1, and α-syn expression levels in the monkey SN. (J–L) Densitometric analysis of TH (J), LRP1 (K), and α-syn (L) expression levels in the SN. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Sham group (unpaired t -test). LRP1: Low-density lipoprotein receptor-related protein 1; PD: Parkinson’s disease; PFF: pre-formed fibril; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Injection, Saline, Immunohistochemistry, Quantitation Assay, Western Blot

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 expression is increased in the nigrostriatal system of a mouse model of PD induced by injection with α-Syn PFFs. (A) Visual gait analysis of walking, gait, and footprint pressure in mice after stereotactic injection with 5 μg of α-Syn PFFs or an equal volume of PBS for 4 weeks ( n = 6). (B) Normal step sequence in mice ( n = 6). (C) Average speed in mice ( n = 6). (D) Tripod support time in mice ( n = 6). (E) Immunohistochemistry staining for TH in the STR. The images in the second row are enlarged images of the areas indicated in black boxes in the upper row. The TH signal intensity in the STR was lower in the PFF group. Scale bars: 200 μm, 40 μm (enlarged). (F) Immunohistochemistry staining for TH in the SN. The images in the second row are enlarged images of the areas indicated in black boxes in the upper row, and the white arrowheads indicate typical TH-positive neurons. There were fewer TH-positive neurons in the SN in the PFF group. Scale bars: 100 μm, 20 μm (enlarged). (G) Quantitative immunohistochemistry density analysis of TH in the STR, compared with the Sham group ( n = 3). (H) Quantitation of the ratio of TH-positive neurons in the SN in the PFF group compared with the Sham group ( n = 3). (I) Western blot analysis of TH, LRP1, and α-Syn expression levels in the mouse STR after injection with 5 μg of α-Syn PFFs or an equal volume of PBS ( n = 3). (J–L) Densitometric analysis of TH (J), LRP1 (K), and α-Syn (L) expression levels in the STR. (M) Western blot analysis of TH, LRP1, and α-Syn expression levels in the mouse SN ( n = 3). (N–P) Densitometric analysis of TH (N), LRP1 (O), and α-Syn (P) expression levels in the SN ( n = 3). Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . Sham group (unpaired t -test). LRP1: Low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PD: Parkinson’s disease; PFF: pre-formed fibril; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-Syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Injection, Sequencing, Immunohistochemistry, Staining, Quantitation Assay, Western Blot

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: Exogenous α-syn PFFs upregulate LRP1 expression in PC12 cells. (A) Viability of PC12 cells incubated with different doses (0, 5, 10, 20, or 50 µg/mL) of α-Syn PFFs for 24 hours. ** P < 0.01, *** P < 0.001, vs . 0 µg/mL group. (B) Western blot analysis of LRP1 and α-syn expression levels in PC12 cells. (C, D) Densitometric analysis of LRP1 (C) and α-syn (D) expression levels. (E) Immunofluorescence analysis of LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) expression. The LRP1 and α-syn signal intensities were stronger in PFF group than in Con group. Scale bars: 40 μm. (F, G) Quantitative immunofluorescence intensity analysis of LRP1 (F) and α-syn (G). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . Con group; # P < 0.05, ## P < 0.01, vs. monomer group. Data are expressed as mean ± SD ( n = 3). Con: Control; DAPI: 4′,6-diamidino-2-phenylindole; PFF: pre-formed fibril; LRP1: low-density lipoprotein receptor-related protein 1; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Expressing, Incubation, Western Blot, Immunofluorescence, Control

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 knockdown rescues the dopaminergic damage induced by exogenous α-syn PFFs. (A, B) Western blot analysis (A) and densitometric analysis (B) of TH expression levels in the STR of mice treated with PFFs. (C, D) Western blot analysis (C) and densitometric analysis (D) of TH expression levels in the SN of mice treated with PFFs. (E) Immunohistochemistry staining for TH in the STR of mice treated with PFFs and LRP1 siRNA. The decrease in relative TH intensity in STR observed in the PFF group was rescued by LRP1 siRNA treatment. The images in the second row are enlarged images of the areas indicated by black boxes in the upper row. Scale bars: 200 μm, 40 μm (enlarged). (F) Quantitative immunohistochemistry density analysis of TH in the STR. (G) Immunohistochemistry analysis of TH in the SN of mice treated with PFFs. The decreased ratio of TH-positive neurons in the SN of the Scramble + PFF group (PFF group) was rescued by LRP1 siRNA treatment. The images in the second row are enlarged images of the areas indicated by black boxes in the upper row, and the white arrowheads indicate typical TH-positive neurons. Scale bars: 100 μm, 20 μm (enlarged). (H) Quantitation of the ratio of TH-positive neurons in the SN compared with the PBS group. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001, vs. Scramble + PBS group (PBS group); # P < 0.05, ## P < 0.01, vs. scramble + PFF group (PFF group). LRP1: Low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PFF: pre-formed fibril; siRNA: small interfering RNA; SN: substantia nigra; STR: striatum; TH: tyrosine hydroxylase; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Knockdown, Western Blot, Expressing, Immunohistochemistry, Staining, Quantitation Assay, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 suppression reduces the transmission of exogenous α-syn in vivo . (A) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) in the mouse STR after stereotactic injection with α-syn PFFs for 6 weeks with or without LRP1 knockdown. The increase in the relative fluorescence intensity of LRP1 and α-syn in the STR of PFFs-treated mice was rescued by LRP1 siRNA treatment. Scale bars: 100 μm. (B, C) Quantitative immunofluorescence intensity of LRP1 (B) and α-syn (C) in mouse STR. (D) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue) in the SN of mice after stereotactic injection with α-syn PFFs for 6 weeks with or without LRP1 knockdown. The increase in the relative fluorescence intensity of LRP1 and α-syn in the SN of mice treated with PFFs was rescued by LRP1 siRNA treatment. The white arrowheads indicate typical cells exhibiting α-syn and LRP1 expression. Scale bars: 100 μm. (E, F) Quantitative immunofluorescence intensity of LRP1 (E) and α-syn (F) in the mouse SN. (G) Western blot analysis of LRP1 and α-syn expression levels in the mouse STR. (H, I) Densitometric analysis of LRP1 (H) and α-syn (I) expression levels in the mouse STR. (J) Western blot analysis of LRP1 and α-syn expression levels in the mouse SN. (K, L) Densitometric analysis of LRP1 (K) and α-syn (L) expression levels in the mouse SN. Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, vs. Scramble + PBS group (PBS group); # P < 0.05, ## P < 0.01, vs . Scramble + PFF group (PFF group). DAPI: 4′,6-Diamidino-2-phenylindole; LRP1: low-density lipoprotein receptor-related protein 1; PBS: phosphate buffered solution; PFF: pre-formed fibril; siRNA: small interfering RNA; SN: substantia nigra; STR: striatum; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Transmission Assay, In Vivo, Immunofluorescence, Staining, Injection, Knockdown, Fluorescence, Expressing, Western Blot, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: LRP1 mediates the uptake of α-syn PFFs by PC12 cells. (A) Western blot analysis of LRP1 and α-syn expression levels in PC12 cells with or without LRP1 knockdown (10 nM siRNA) after incubation with 10 µg/mL α-syn PFFs for 24 hours. (B, C) Densitometric analysis of LRP1 (B) and α-syn (C) expression. (D) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-syn (red, Alexa Fluor 594), and DAPI (blue). The increase in the relative fluorescence intensity levels of LRP1 and α-syn in the PFF group was rescued by LRP1 siRNA treatment. Scale bars: 40 μm. (E, F) Quantitative immunofluorescence intensity analysis of LRP1 (E) and α-syn (F). Data are expressed as mean ± SD ( n = 3). ** P < 0.01, *** P < 0.001, **** P < 0.0001, vs . Con group; # P < 0.05, ## P < 0.01, #### P < 0.0001, vs . PFF/– group. Con: Control; DAPI: 4′, 6-diamidino-2-phenylindole; PFF: pre-formed fibril; LRP1: low-density lipoprotein receptor-related protein 1; siRNA: small interfering RNA; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Western Blot, Expressing, Knockdown, Incubation, Immunofluorescence, Staining, Fluorescence, Control, Small Interfering RNA

Journal: Neural Regeneration Research

Article Title: Low-density lipoprotein receptor–related protein 1 mediates α-synuclein transmission from the striatum to the substantia nigra in animal models of Parkinson’s disease

doi: 10.4103/NRR.NRR-D-23-01965

Figure Lengend Snippet: Lysine residues in the α-syn N-terminus are vital for LRP1-mediated α-Syn internalization. (A) Heatmap of amino acids in different α-syn domains. (B) Western blot analysis of LRP1 and α-Syn levels in PC12 cells after addition of α-syn PFFs (10 µg/mL) with or without capping of lysine residues for 24 hours. (C, D) Densitometric analysis of LRP1 (C) and α-syn (D) expression levels. (E) Immunofluorescence staining for LRP1 (green, Alexa Fluor 488), α-Syn (red, Alexa Fluor 594), and DAPI (blue). The increase in the relative fluorescence intensity of LRP1 and α-syn in the PFF group was rescued by lysine capping of α-syn. Scale bars: 40 μm. (F, G) Quantitative immunofluorescence intensity analysis of LRP1 (F) and α-syn (G). Data are expressed as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, **** P < 0.0001, vs. Con group; # P < 0.05, ## P < 0.01, vs . PFF group. Con: Control; DAPI: 4′,6-diamidino-2-phenylindole; LRP1: low-density lipoprotein receptor-related protein 1; NHS: sulfo-NHS acetate; PFF: pre-formed fibril; α-syn: α-synuclein.

Article Snippet: α-Syn PFFs (SPR-322, StressMarq, Victoria, Canada) were sonicated with a handheld probe (VCX130, SONICS & MATERIALS, Newtown, CT, USA) as follows: amplitude 30% and time 15 seconds (pulse on 3 seconds, pulse off 6 seconds) (Volpicelli-Daley et al., 2014; Sun et al., 2022b).

Techniques: Western Blot, Expressing, Immunofluorescence, Staining, Fluorescence, Control

Journal: bioRxiv

Article Title: Structural mechanism of Necrocide 1 activation of human TRPM4 that triggers necrosis by sodium overload

doi: 10.64898/2026.01.28.702369

Figure Lengend Snippet: a Superposition of the hTRPM4 structures in the NC1-bound and apo closed (PDB:9MTA) states with the front subunits highlighted in cyan and orange, respectively. b Local conformational changes at the S1-S4 domain between NC1-bound (cyan) and apo closed (orange) hTRPM4. Only S3-S4 and the neighboring S5 (marked with a single quotation mark) are displayed for clarity. Red arrows mark the major movements upon NC1 binding. Key residues are shown in stick representation. c Structural comparison of the hTRPM4 ion conduction pore in the apo closed (orange), NC1-bound (cyan), and NC1/PI(4,5)P 2 -bound (navy blue) states. Gating residues I1040 and S1044 are shown in stick representation. The front and back subunits are removed for clarity. d Pore radius of hTRPM4 along the central axis in the apo closed, NC1-bound, and NC1/PI(4,5)P 2 -bound structures. e F935’-F910 contact interface in the NC1-bound, apo closed, Ca 2+ -bound desensitized (PDB:9MT8), and Ca 2+ /PI(4,5)P 2 -bound open (PDB:9MRT) states. The EM density maps for F910 and F935’ are shown in grey surface contoured at 0.23 in ChimeraX.

Article Snippet: Therefore, the water-soluble short-chain synthetic PI(4,5)P 2 diC8 (Phosphatidylinositol 4,5-bisphosphate diC8, Echelon Bioscience) was used in the functional experiments as needed.

Techniques: Binding Assay, Comparison

Journal: bioRxiv

Article Title: Structural mechanism of Necrocide 1 activation of human TRPM4 that triggers necrosis by sodium overload

doi: 10.64898/2026.01.28.702369

Figure Lengend Snippet: a Macroscopic currents of hTRPM4-expressing HEK293 cells at ±100 mV in an inside-out patch with the presence or absence of Ca 2+ , NC1, and PI(4,5)P 2 diC8 in the bath (cytosolic). NC1 and PI(4,5)P 2 diC8 were introduced after Ca 2+ activation and desensitization due to Ca 2+ -induced membrane PI(4,5)P 2 depletion. A-E mark the time points of hTRPM4 in various states: A, apo closed state; B, initial Ca 2+ -activated state before desensitization; C, Ca 2+ -bound desensitized state due to PI(4,5)P 2 depletion; D, closed state in the presence of NC1; E, NC1/PI(4,5)P 2 -activated state. The panel on the right depicts sample I-V curves corresponding to A-E time points. b Similar recordings as a except that PI(4,5)P 2 diC8 was introduced before NC1, and the time point D marks the closed state in the presence of PI(4,5)P 2 diC8. The panel on the right depicts sample I-V curves corresponding to A-E time points. c Sample I-V curves of wild-type hTRPM4 and its alanine substitutions at the PI(4,5)P 2 -binding site recorded in whole-cell patches with 10 µM NC1 in the bath (extracellular). d Outward and inward current densities of NC1-activated wild-type hTRPM4 and its mutants recorded at ±100 mV in whole-cell patches with 10 µM NC1 in the bath (extracellular). Bars represent mean ± SEM of n=5 independent replicates. e Responsiveness of HeLa cells to NC1-induced cell death when expressing wild-type hTRPM4 or its PI(4,5)P 2 site mutants. Data points represent mean ± SD of n = 3 independent replicates. Over 50 cells were counted in each replicate.

Article Snippet: Therefore, the water-soluble short-chain synthetic PI(4,5)P 2 diC8 (Phosphatidylinositol 4,5-bisphosphate diC8, Echelon Bioscience) was used in the functional experiments as needed.

Techniques: Expressing, Activation Assay, Membrane, Binding Assay

Journal: bioRxiv

Article Title: Structural mechanism of Necrocide 1 activation of human TRPM4 that triggers necrosis by sodium overload

doi: 10.64898/2026.01.28.702369

Figure Lengend Snippet: a Superposition of the NC1/PI(4,5)P 2 -bound and Ca 2+ /PI(4,5)P 2 -bound (PDB:9MRT) hTRPM4 open structures with the front subunits highlighted in navy blue and pink, respectively. NC1 and PI(4,5)P 2 are depicted in stick representation, and Ca 2+ is shown as a green sphere. b Zoomed-in view of the ligand binding sites in the NC1/PI(4,5)P 2 -bound structure. Ca 2+ is represented as a green sphere. NC1, PI(4,5)P 2, and ligand-interacting residues are shown in stick representation. Density (grey surface) is contoured at 0.5 for NC1 and Ca 2+ and at 0.45 for PI(4,5)P 2 in ChimeraX. c Superposition of the NC1/PI(4,5)P 2 -bound open and NC1-bound hTRPM4 structures with the front subunits highlighted in navy blue and cyan, respectively. d Enlarged view of the conformational changes at the transmembrane region between the NC1/PI(4,5)P 2 -bound open (navy blue) and NC1-bound (cyan) structures. The S4 helix from the neighboring subunit (S4’) is also shown. The inset shows a close-up highlighting the coupling between F935 and neighboring F910’ in the NC1/PI(4,5)P 2 -bound structure that leads to the movement (red arrow) at the S4-S5 linker and S5 and channel opening. Density for F935 and F910’ is shown in a grey surface and contoured at 0.55 in ChimeraX. e Conformational changes at the cytosolic MHR domains between the NC1/PI(4,5)P 2 -bound (navy blue) and NC1-bound (cyan) structures. Red arrows mark the upward swing of the cytosolic MHR domains and the rotation of MHRs 1 and 2.

Article Snippet: Therefore, the water-soluble short-chain synthetic PI(4,5)P 2 diC8 (Phosphatidylinositol 4,5-bisphosphate diC8, Echelon Bioscience) was used in the functional experiments as needed.

Techniques: Ligand Binding Assay

Journal: bioRxiv

Article Title: Structural mechanism of Necrocide 1 activation of human TRPM4 that triggers necrosis by sodium overload

doi: 10.64898/2026.01.28.702369

Figure Lengend Snippet: a Superposition of the NC1/PI(4,5)P 2 -bound (brown) and apo closed (PDB:6BCJ) (green) wild-type mTRPM4 structures along the pore axis, with the front subunits highlighted. NC1 (blue) and PI(4,5)P 2 (yellow) are shown in stick representation. b Zoomed-in view of the NC1 binding site in mTRPM4. NC1 and its interacting residues are shown in stick representation. NC1 density (grey surface) is contoured at 0.23 in ChimeraX. c Conformational changes at S1-S4 and its neighboring pore domain upon NC1 binding to mTRPM4. The red line and red diamond mark the central 4-fold axis. Red arrows indicate the major movements in this region, namely the lateral movement of S1-S4 towards the central axis, the inward movement of the neighboring subunit S5 (S5’), and the outward movement of the neighboring subunit S6 (S6’). d Comparison of the density maps at the pore domain between the apo closed (PDB:6bcj) (green) and NC1/PI(4,5)P 2- bound (brown) mTRPM4 structures. The disordered region in the pore domain of the NC1/PI(4,5)P 2- bound structure is depicted as a grey semi-transparent cartoon. e Structural comparison of the ion conduction pore in the apo closed (green), NC1/PI(4,5)P 2 -bound inactivated (brown) wild-type mTRPM4 and in the NC1/PI(4,5)P 2 -bound open (purple) triple mutant (L900V/R993P/S1060R). Gating residues I1036 and S1040 are shown in stick representation. The front and back subunits were removed for clarity. The panel on the right depicts the pore radius along the central axis in these structures. The dotted line illustrates the disordered filter region in the NC1/PI(4,5)P 2 -bound wild-type mTRPM4. f Structure of the NC1/PI(4,5)P 2- bound mTRPM4 triple mutant (L900V/R993P/S1060R) with the front subunit highlighted in purple. NC1 and PI(4,5)P 2 are shown in stick representation and Ca 2+ as a green sphere. g Structural comparison at S1060 region between NC1/PI(4,5)P 2- bound wild-type mTRPM4 (light brown) and its triple mutant (L900V/R993P/S1060R, light purple). PI(4,5)P 2 and key residues important for stabilizing S1-S4 in the mutant are shown in stick representation. The wild-type S1060 side chain is also shown for comparison.

Article Snippet: Therefore, the water-soluble short-chain synthetic PI(4,5)P 2 diC8 (Phosphatidylinositol 4,5-bisphosphate diC8, Echelon Bioscience) was used in the functional experiments as needed.

Techniques: Binding Assay, Comparison, Mutagenesis